This application is a 371 of PCT/FR93/01202 Dec. 7, 1993.
The present invention relates to cells modified with respect to the catabolism of betaine, to their preparation and their use, especially for the improved production of metabolites and/or enzymes. The invention also relates to DNA fragments carrying genes for the catabolism of betaine.
Glycine betaine (N,N,N-trimethylglycine) is generally known for its osmoprotective properties, which confer on bacteria tolerance to osmotic stress (Csonka, 1989). To explain the origin of this property, it has been proposed that the molecular effects of glycine betaine on the activity of water and on the osmotic pressure of the cytoplasm in Escherichia coli were more important than those of the solutes which it replaces (Cayley et al, 1992). Furthermore, in addition to its osmoprotective potentials, it has been described that glycine betaine could also promote the production of enzymes (JP 8,260,709) or of metabolites, such as amino acids (Patent JP 202703); antibiotics (Patent AU 825513) and vitamins (White et al, 1971). However, most bacteria except cyanobacteria and other CO2-fixing prokaryotes do not synthesize glycine betaine which is mainly synthesized by plants. It should therefore be added to production media in fermenters, which generates an additional cost in an industrial process. The present invention provides a solution to this problem.
The applicant has indeed demonstrated that it is possible, by modifying the catabolism of betaine of cells, especially by genetic means, to potentiate the effect of this compound on the production of enzymes or of metabolites without affecting the rate of growth of cells, their viability and the like, under industrial fermentation conditions. The applicant has also identified, isolated and characterized DNA fragments containing genes involved in the catabolism of betaine, which make it possible in particular to prepare cells specifically modified with respect to the catabolism of betaine, and whose modifications are segregationally stable and nonreversible. These fragments also make it possible to stimulate the catabolism of betaine by amplification of the appropriate enzymatic activities. The present invention therefore makes it possible to potentiate the effects of betaine and, thereby, to use this compound economically in industrial fermentation processes.
A first subject of the invention therefore relates to a modified cell exhibiting at least one modification with respect to a gene involved in the catabolism of betaine.
In a first embodiment, the term modified cell designates more particularly any cell having a substitution and/or a deletion and/or an insertion of one or more bases in the considered gene(s) and degrading betaine less rapidly. Such modifications can be obtained in vitro (on isolated DNA fragments carrying genes for the catabolism of betaine) or in situ, for example, by means of genetic engineering techniques, or alternatively by exposing the said cells to a treatment by means of mutagenic agents.
As mutagenic agents, there may be mentioned for example physical agents such as energetic radiation (X-, g- or ultraviolet rays end the like) or chemical agents capable of reacting with various functional groups of the bases of DNA, and for example alkylating agents [ethyl methane-sulphonate (EMS), N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine, N-nitroquinoline 1-oxide (NQO)], dialkylating agents, intercalating agents and the like.
Deletion is understood to mean the removal of all or part of the gene considered. This may especially be a portion of the coding region and/or of all or part of the promoter region for transcription.
The genetic modifications can also be obtained by gene disruption, for example according to the procedure initially described by Rothstein (1983). In this case, all or part of the gene is preferably perturbed so as to allow the replacement, by homologous recombination, of the wild-type genomic sequence by a nonfunctional or mutant sequence prepared in vitro.
The said modification(s) may be located in the coding portion of the gene or in the regions responsible for the expression end/or transcriptional regulation of the said genes. The (total or partial) incapacity of the said cells to degrade betaine can manifest itself either by the production of inactive enzymes because of structural or conformational modifications, or by the absence of production, or by the production of enzymes having an impaired activity, or alternatively by the production of natural enzymes at en attenuated level or according to a desired mode of regulation.
Moreover, certain modifications such as point mutations are by nature capable of being corrected or attenuated by cellular mechanisms, for example during the replication of DNA preceding cell division. Such genetic modifications are, in this case, of limited interest at the industrial level since the phenotypic properties resulting therefrom are not perfectly stable. According to the present invention, it is now possible, by virtue of the identification of DNA fragments carrying genes for the catabolism of betaine, to prepare modified cells in which the said modification(s) are segregationally stable and/or nonreversible. The cells exhibiting such modifications are particularly advantageous as cellular host for the production of metabolites end/or enzymes.
In another particular embodiment, the modified cells of the invention are cells in which at least one gene involved in the catabolism of betaine is amplified, and which as a result degrade betaine more rapidly.
The amplification can be obtained by introducing a DNA fragment carrying a gene for the catabolism of betaine into the cell. This fragment is preferably part of a vector, which my be an autonomously replicating vector or an integrative vector. Moreover, the DNA fragment may be homologous or heterologous in relation to the modified cell, that is to say that the amplified gene(s) may be genes from the said cell or genes obtained from other cellular sources and encoding an activity of the same type. The choice of the vector and of the origin of the amplified fragment depends on the cells considered and the applications envisaged. The DNA fragment may be introduced into the cells by any method allowing the introduction of a foreign DNA into a cell. This may be in particular transformation, electroporation, conjugation, protoplast fusion, or any other techniques known to persons skilled in the art.
According to studies carried out essentially in Rizobium meliloti, the degradation of glycine betaine is performed on media of low osmolarity, by three successive demethylations (see FIG. 1). The first stage is catalysed by betaine homocysteine methyltransferase E.C. 2.1.1.5. and leads to dimethylglycine; the second is catalysed by dimethylglycine dehydrogenase E.C. 1.5.99.2 and generates momomethylglycine or sarcosine; finally the third is catalysed by sarcosine dehydrogenase E.C. 1.5.99.1 and the product of the reaction is glycine (Smith et al, 1988).
Preferably, the modifications exhibited by the cells of the invention affect one of the fist two stages of the catabolism of betaine, or optionally the two first stages simultaneously.
Preferably, the cells of the invention are cells which product metabolites and/or enzymes. In this respect, they may also be recombinant cells which product metabolites and/or enzymes, that is to say cells modified by recombinant DNA techniques so as to improve their production capacity (cf. especially WO 91/11518, EP 346000). Still more preferably, the cells of the invention are chosen from cells of the genus Pseudomonas, Streptomyces, actinomycetes, Propionibacterium, Corynabacterium, Bacillus, Escherichia, Salmonella, Rhizobium, Agrobacterium, Rhodopseudomonas, Xanthomonas, Clostridium end Methanobacterium.
Another aspect of the invention relates to a process for preparing cells exhibiting a modification of at least one gene involved in the catabolism of betaine which are capable of being used under industrial fermentation conditions.
The present invention in effect describes the identification, isolation and characterization of DNA fragments containing genes involved in the catabolism of betaine. These fragments now make it possible to prepare cells which are specifically modified with respect to the catabolism of betaine. It is indeed possible to modify in vitro the fragments described so as to make them nonfunctional and to reintroduce them into a given cell, in which they will become substituted by double homologous recombination for the corresponding functional genomic copy. It is also possible, on the basis of the fragments thus isolated, to prepare probes which will become integrated into the genome of a desired cell, specifically in the corresponding gene.
More particularly, the process of the invention consists in replacing the considered chromosomal gene(s) by versions modified in vitro.
Another subject of the invention relates to a DNA fragment carrying at least one gene for the catabolism of betaine.
More particularly, the present invention is illustrated by the isolation and characterization of DNA fragments from Pseudomonas denitrificans which complement the mutants blocked in the catabolism of glycine betaine into dimethylglycine and those blocked in the catabolism of dimethylglycine into sarcosine, that is to say the DNA fragments containing genes involved in the degradation of glycine betaine into dimethylglycine and of dimethylglycine into sarcosine. The DNA fragments according to the invention were isolated from a Pseudomonas denitrificans strain SC510, which is derived from the strain MB580 (U.S. Pat. No. 3,018,225). These fragments were obtained
(i) preparation of mutants blocked in the catabolism of betaine. Various techniques already mentioned above can be used to this end. The selection of the is performed by culturing on an appropriate medium and assaying, according to conventional techniques for persons skilled in the art, betaine or products of its acid from a microorganism capable of catabolizing catabolism.
(ii) complementation of three mutants with nucleic betaine,
(iii) selection of the complemented mutants, then isolation and characterization of the nucleic acid having allowed this complementation, which therefore carries genes for the catabolism of betaine.
It is clear that, from the DNA fragments identified and isolated in the present application, persons skilled in the art can, especially by hybridization experiments, isolate and clone genes for the catabolism of betaine from other cellular sources.
More preferably, the gene in question is therefore the one encoding betaine-homocysteine methyltransferase, on which gene a modification according to the invention induces, in the cell, a decrease in the betaine-homocysteine methyltransferase activity. Preferably still, the gene in question is the one encoding dimethylglycine dehydrogenase, on which a modification according to the invention induces, in the cell, a decrease in the dimethylglycine dehydrogenase activity.
Another subject of the invention relates to an improved process for producing metabolites or enzymes by culturing a cell which produces the said metabolite or enzyme, modified with respect to its catabolism of betaine, under production conditions, and then recovering the said metabolite or enzyme.
According to a first embodiment, the producing cell exhibits at least one modification with respect to a gene involved in the catabolism of betaine and degrades betaine less rapidly.
Preferably, according to the first embodiment, the modification(s) are segregationally stable and/or nonreversible.
In a preferred variant of the invention, the modification(s) are mutational deletions and/or insertions.
According to a second embodiment, at least one gene of the producing cell, which is involved in the catabolism of betaine, is amplified and the said cell degrades betaine more rapidly.
The process of the invention is particularly suitable for the production of metabolites such as amino acids, vitamins, antibiotics, their derivatives or their precursors.
The process of the invention can especially allow the improved production of cobalamin, and preferably of vitamin B12.
The present invention is supplemented by the following examples which should be considered as illustrative and nonlimiting.
The methods conventionally used in molecular biology such as preparative plasmid DNA extractions, plasmid DNA centrifugation in a caesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, phenol or phenol-chloroform extraction of proteins, DNA precipitation in salt medium with ethanol or isopropanol,. transformation in Escherichia coli and the like, are well known to persons skilled in the art and are widely described in the literature [Maniatis T. et al., xe2x80x9cMolecular Cloning, a Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F. M. et al. (eds), xe2x80x9cCurrent Protocols in Molecular Biologyxe2x80x9d, John Wiley and Sons, New York, 1987].
The restriction enzymes were produced by New England Biolabs (Biolabs), or Pharmacia and are used according to the recommendations of the suppliers.
The pBR322 and pUC type plasmids are of commercial origin (Bethesda Research Laboratories).
For the ligations, the DNA fragments are separated according to their size by electrophoresis on agarose or acrylamide gels, exctracted with phenol or with a phenol/chloroform mixture, precipitated with ethanol and then incubated in the presence of T4 phage DNA ligase (Boehringer) according to the recommendations of the supplier.
The filling of the protruding 5xe2x80x2 ends is performed by the Klenow fragment of DNA polymerase I of E. coli according to the specifications of the supplier. The destruction of the protruding 3xe2x80x2 ends is performed in the presence of T4 phase DNA polymerase used according to the recommendations of the manufacturer. The destruction of the protruding 5xe2x80x2 ends is performed by a controlled treatment with S1 nuclease.
Site-directed mutagenesis in vitro by synthetic oligodeoxynucleotides is performed according to the method developed by Taylor et al. [Nucleic Acids Res. 13 (1985) 8749-8764].
The enzymatic amplification of the DNA fragments by the so-called PCR technique [Polymerase-catalysed Chain Reaction, Saiki R. K. et al., Science 230 (1985) 1350-1354; Mullis K. B. et Faloona F. A., Meth. Enzyme. 155 (1987) 335-350] is performed using a xe2x80x9cDNA thermal cyclerxe2x80x9d (Perkin Elmer Cetus) according to the specifications of the manufacturer.
The verification of the nucleotide sequences is performed by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5463-5467].
Betaine: glycine betaine or N,N,N-trimethylglycine
HPLC: high-pressure liquid chromatography
DMG: dimethylglycine
kb: kilobase